Peptido-mimetic compounds containing RGD sequence useful as integrin inhibitors

ABSTRACT

The present invention discloses compounds of formula (I)  
                 
 
     wherein n is the number 0, 1 or 2. There are also disclosed processes for the preparation of said compounds, together with methods for treating pathologies related to an altered α v β 3  integrin-mediated cell attachment, in particular wherein the inhibition of angiogenesis is desired, for example in tumors, also associated with metastasis.

1. The present invention relates to cyclic peptidomimetic compounds, inparticular to cyclic peptidomimetic compounds having azabicycloalkanestructure and containing the RGD (Arg-Gly-Asp) sequence. Said compoundshave inhibiting action on α_(v)β₃-receptor of the integrin family. Thecompounds of the present invention are endowed with antiangiogenicproperties, hence are useful as medicaments, preferably for thetreatment of tumors.

BACKGROUND OF THE INVENTION

2. The first molecule with antiangiogenic activity was discovered in1975 by Henry Brem and Judah Folkman in cartilaginous tissues.

3. In the 80s it was found that interferon (α/β) is effective ininhibiting tumor angiogenesis.

4. In 1998, it was widely published, also in the media, that angiostatinand endostatin discovered by J. Folkman at Harvard Medicinal School andBoston Children's Hospital were giving very encouraging results intuxnor treatment.

5. To-date, about 30 molecules are tested in clinical trials (PhaseI-III).

6. Of these 30 molecules, only two drugs, of which one is an antibody,are in clinical trials for their activity in inhibiting endothelialspecific integrins.

7. It is calculated that only in the USA, about 9 million patients couldbenefit from an antiangiogenic therapy.

8. Recently, FDA has approved clinical trials for the combination ofIL-10 with Thalidomide and Methoxyestradiol.

9. Angiogenesis is intended as the formation of new capillary bloodvessels. This natural phenomenon is involved both in physiologicalprocesses, as reproduction, and in pathological occurrences, as woundhealing, arthritis and tumor vascularization.

10. A number of growth factors have been identified as capable ofpromoting angiogenesis, through direct induction of proliferation and/orchemiotaxis of endothelial cells. Other factors, instead, actindirectly, by stimulating other cell types (mast cells, macrophages),which, on their turn, produce angiogenic factors. The presence of growthfactors, such as bFGF and VEGF, near a resting capillary net, suggestedthat angiogenesis might be the outcome of an unbalance between pro- andanti-angiogenic factors.

11. In the last years, it was reported that tumor growth and metastasisformation is strictly dependent on the development of new vesselscapable of vascularizing the tumor mass.

12. Antiangiogenic tumor therapy is strongly desired by physicians forthe following reasons:

13. specificity: tumor tieovascularization is the target;

14. bioavailability: the antiangiogenic agent is targeted towardendothelial cells, easily reached without the well-known problems ofchemotherapy, which is directed on the tumor cell;

15. chemoresistance; this is the most striking advantage, in fact,endothelial cells are genetically stable and it is quite difficult toobserve drug resistance;

16. angiogenic blockade avoids metastatic cells to diffuse through bloodcirculation;

17. apoptosis: blocking angiogenesis makes tumor cell suffer from oxygenand nutrition lack, thus inducing apoptosis;

18. antiangiogenic therapy does not give rise to side effects typical ofchemotherapy.

19. The endogenous pro-angiogenic factors to date known are acid/basicFibroblast Growth factor (a/bFGF) and Vascular Endothelial Growth Factor(VEGF), and its subtype B and C, Angiogenin, Endothelial Growth Factor(EGF), Platelet derived-Endothelial Cell Growth Factor (PD-ECGF),Transforming Growth Factor-α (TGF-α), Transforming Growth Factor-β(TGF-β), Tumor Necrosis Factor-α (TNF-α).

20. Retinoids are tested as potential antiangiogenic agents.

21. Some PK-C inhibitors, such as Calphostin-C, phorbol esters andStaurosporin, can block angiogenesis, either partially or totally.

22. Integrins are a class of receptors involved in the mechanism of celladhesion and alterations in the function of these receptors areresponsible in the occurrence of a number of pathologic manifestations,for example embryogenic development, blood coagulation, osteoporosis,acute renal failure, retinopathy, cancer, in particular metastasis.Among the molecular targets involved in angiogenesis, α_(v)β₃ integrinsplay an important role in adhesion, motility, growth and differentiationof endothelial cells. α_(v)β₃ integrins bind the RGD sequence(Arg-Gly-Asp), which constitutes the recognition domain of differentproteins, such as laminin, fibronectin and vitronectin. The role of RGDsequence is described, for example, in Grant et al., J. Cell Physiology,1992, Saiki et al., Jpn. J. Cancer Res. 81; 668-75. Carron et al, 1998,Cancer Res. 1; 58(9):1930-5 disclosed an RGD-containing tripeptide,named SC-68448, capable of inhibiting the binding between αvβ3 integrinwith vitronectin (IC₅₀=1 nM), Other works (Sheu et al., 1197, BBA;1336(3):445-54 - Buckle at al., 1999, Nature 397:534-9) showed that RGDpeptides can diffuse through the cell membrane and bind to the proteincaspase-3, inducing apoptosis.

23. Therefore, RGD sequence is the basis for developing antagonists ofthe different integrins. To date, the reasons for which in many cases ahigh selectivity for certain integrins is observed is not quite clear,although a different conformation of the RGD sequence can be taken as anexplanation. Recent data demonstrated that this sequence is ofteninserted into a type II-β-turn between two β-sheets extending from thecore of the protein.

24. Thus the problem to provide substances having high selectivitytoward integrins has not been fully satisfied yet.

25. There is a structural constraint to this research, namely, the RGDsequence must be kept unaltered, since it is well known that anymodification to this sequence implies a loss of activity.

26. To find the correct structure that can block the molecule in aprecise reverse-turn conformation, inducing a β-turn geometry, is verycritical.

27. It is well known that the α_(v)β₃-receptor, a member of the integrinfamily, is implicated in angiogenesis and in human tumor metastasis.

28. Metastasis of several tumor cell lines as well as tumor-inducedangiogenesis can be inhibited by antibodies or small, synthetic peptidesacting as ligands for these receptors (Friedlander et al.: Science 1995,270, 1500-1502.

29. In order to have an inhibiting property, all the peptides mustcontain the Arg-Gly-Asp (RGD) sequence. Notwithstanding this RGDsequence, a high substrate specificity is present, due to differentconformations of the RGD sequence in different matrix proteins(Ruoshlati et al. Science 1987, 238, 491-497). This flexibility ofparticular RGD portion is an obstacle to the determination of thebioactive conformation to be used in the widespread struture-activitydrug design.

30. A solution was provided by Haubner et al. (J. Am. Chem. Soc. 1996,118, 7881-7891) by inserting the RGD sequence in cyclic, rigid peptidestructure. Spatial screening led to the highly active first-generationpeptide c(RGDfV) (cyclic Arg-Gly-Asp-D-Phe-Val; WO97/06791), which showsa βII'/γ-turn arrangement. A reduction of the flexibility is a technicalgoal to be achieved in order to obtain antagonists of integrins. Due tothe width of the integrin family and to the number of differentphysiological activities of said integrins, it is highly desired toobtain active agents having highly selective inhibiting action.

31. A solution proposed in the art was to introduce in thepeptidomimetic structure a rigid building block (turn mimetics).

32. Despite different tentatives and a number of structures proposed,Haubner et al (J. Am. Chem. Soc. 1996, 118, 7881-7891), identified anRGD“spiro” structure capable of providing the desired βII'/γ-turnarrangement. Actually, four different structures are enabled in thiswork: an (S)-proline derivative, an (R)-proline derivative, athiazabicyclo structure and a diaza-spiro-bicyclic structure.Non-homogeneous results were obtained. The Spiro structure was the onlyone able to adopt a βII'/γ-turn conformation, but lacks of biologicalactivity, The (S)-proline is very active, but less selective. The(R)-proline is active and selective. The thiazabicyclo-structure isactive, but has the disadvantage to be less selective.

33. WO91/15515 discloses cyclic peptides, also containing the RGDsequence, useful for treating thrombosis, through the selectiveinhibition of the platelet aggregation receptor GPIIb/IIa.

34. WO92/17492 discloses cyclic peptides, also containing the RGDsequence, useful for treating thrombosis, through the selectiveinhibition of the platelet aggregation receptor GPIIb/IIa. Thesepeptides contain also a positively charged nitrogen containing exocyclicmoiety stably bonded to the cyclic peptide through a carbonyl. Nobeta-turns are contained in these structures.

35. WO94/29349 discloses a long peptide containing a -Cys-S-S-Cys-cyclic portion for the treatment of a venous or arterial thromboticcondition. This trifunctional peptide combines both catalytic and anionbinding exosite inhibition of thrombin with GP IIb/IIIa receptorinhibition.

36. Other peptides active in treating thrombosis are disclosed inWO95/00544.

37. WO97/06791 discloses the use of c(RGDfV) as selective inhibitor ofα_(v)/γ₅ and useful as inhibitor of angiogenesis

38. WO97/08203 discloses circular RGD-containing peptides, whichcomprise the motif (/P)DD(G/L)(W/L)(W/L/M).

39. U.S. Pat. Nos. 5,767,071 and 5,780,426 disclose non-RGD amino acidcyclic peptides binding α_(v)/γ₃ integrin receptor.

40. U.S. Pat. No. 5,766,591 discloses RGD-peptides for inhibitingα_(v)/γ₃ receptor and useful as antiangiogenesis agents. No beta turnportions are taught.

41. WO98/56407 and WO98/56408 disclose fibronectin antagonists astherapeutic agents and broad-spectrum enhancers of antibiotic therapy.Said fibronectin antagonists bind to a α₅β₁ integrin to the purpose toprevent intracellular invasion by microbial pathogens. Some of theseinhibitors are linear or cyclic peptides containing the RGD structure orantibodies. Integrin antagonists are specifically disclosed for theirselectivity against α₅β₁ integrin. The best of them proved to be(S)-2-[2,4,6-trimethylphenyl)sulfonyl]amino-3-[[7benzyloxycarbonyl-8-(2-pyridinylaminomethyl)-1-oxa-2,7-diazaspiro-[4,4]-non-2-en-3-yl]carbonylamio]propionicacid.

42. U.S. Pat. No. 5,773,412 discloses a method for altering α_(v)β₃integrin receptor-mediated binding of a cell to a matrix, said cellbeing an endothelial or smooth muscle cell, by contacting said cell witha RGD-containing cyclic peptide. Also disclosed there is a method forinhibiting angiogenesis by using this cyclic peptide. The cyclic peptidedisclosed in U.S. Pat. No. 5,773,412 contains at least 6 amino acids andthe RGD sequence is flanked, on the D-side, by a first amino acid whichcan provide a hydrogen bond interaction with an integrin receptor (Asn,Ser or Thr) and a second amino acid, that has the characteristics ofhydrophobicity or conformational constraint (Tic, i.e.1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, Pro, Phe or Ile). Aselection of these peptides are taught as useful for altering thebinding of osteoclasts to a matrix such as bone or for selectivelyaltering integrin receptor binding.

43. It has now been found that cyclic pseudopeptides having an RGDmimetic structure characterized by an azabicycloalkane structure areendowed with selective inhibition of α_(v)β₃ integrin-mediated cellattachment. This activity makes them useful as therapeutical agents, inparticular for treating pathologies due to an altered angiogenesis, forexample tumors.

ABSTRACT OF THE INVENTIONn

44. It is an object of the present invention, compounds of formula (I)

45. wherein n is the number 0, 1 or 2,

46. Asp is the amino acid L-Arginine, Gly is the amino acid Glycine andAsp is the amino acid L-Aspartic acid, and the pharmaceuticallyacceptable salts thereof, their racemates, single to enantiomers andstereoisomers.

47. The compounds of formula (I) are selective inhibitors of α_(v)β₃receptor. Accordingly, they are useful for treating all thosepathologies due to an altered α_(v)β₃ integrin-mediated cell attachment;for example, retinopathies, acute renal failure, osteoporosis, tumors,also associated with metastasis. The compounds of the present inventioncan be considered as antiangiogenesis agents, in particular for thetreatment of tumors, comprising tumors associated with metastasis.

48. Other objects of the present invention are processes for thepreparation of the compounds of formula (I).

49. A further object of the present invention is a method for treating asubject, whether human or animal, suffehng of a tumor, by inducing aninhibition of angiogenesis, in particular for inhibiting or reducing orblocking metastatic proliferation, with the administration of atherapeutic or preventive dose of at least a compound of formula (I).Also objects of the present invention are: a method for selectivelyinhibiting α_(v)β₃ integrin-mediated cell attachment to anRGD-containing ligand, comprising contacting said ligand with aneffective amount of a compound of formula (I); a method for treating asubject suffering from a pathology related to an altered α_(v)β₃integrin-mediated cell attachment comprising administering to saidsubject a compound of formula (I); said pathologies being for exampleretinopathy, acute renal failure, osteoporosis.

50. From the industrial application point of view, the present inventionalso comprises pharmaceutical compositions comprising an effective doseof at least a compound of formula (I) in admixture with pharmaceuticallyacceptable vehicles and/or excipients.

51. The present invention shall be disclosed in detail in the foregoingalso by means of examples and figures, wherein, in the figures:

52.FIG. 1 represents, in an exemplary way, the general synthesis of thelactams;

53.FIG. 2 represents a preferred embodiment of the synthesis of6,5-fused “cis” lactams;

54.FIG. 3 represents a preferred embodiment of stereoselectivehydrogenation with chiral phosphine-Rh catalyst;

55.FIG. 4 represents a preferred embodiment of the synthesis of7,5-fused “cis” lactams;

56.FIG. 5 represents a preferred embodiment of the synthesis of5,5-fused “cis” lactams;

57.FIG. 6 represents another preferred embodiment of the synthesis of5,5-fused “cis” lactams;

58.FIG. 7 represents a preferred embodiment of the synthesis of6,5-fused “trans” lactams;

59.FIG. 8 represents a preferred embodiment of the synthesis of7,5-fused “trans” lactams.

DETAILED DESCRIPTION OF THE INVENTION

60. In its broadest aspects, the present invention relates to compoundsof the above formula (I).

61. The compounds of formula (I) are peptidomimetics containing an RGDsequence. Said compounds can be seen as formed by an azabicycloalkanescaffold and an RGD sequence.

62. For sake of clarity, in formula (I), there is a variable part, givenby the different values of n, and a fixed part, given by the RGDsequence. When n is 0, the scaffold is referred to as 5,5azabicycloalkane, when n is 1, the scaffold is referred to as 6,5azabicycloalkane and when n is 2, the scaffold is referred to as 7,5azabicycloalkane. The bonds written in formula (I) as a wavy linerepresents a stereo bond, which can be either above the plane of thepage (thick bond) either below the plane of the page (thin bond). Thecompounds of formula (I) can exist in different stereoisomers, accordingto the orientation of the wavy bond. In the following table there arerepresented the preferred compounds of formula (I):

63. Within the boundaries of the present invention, there is disclosed aprocess for the preparation of the compounds of formula (I), comprisingthe following steps:

64. a) Horner-Emmons olefination of a compound of formula (II)

65. wherein

66. R is a lower allyl residue;

67. R₁ is a suitable nitrogen protecting group, to give a compound offormula (III);

68. wherein R₃ is a suitable nitrogen protecting group, R₄ is a loweralkyl residue;

69. b) hydrogenation of said compound of formula (III) and cyclisation;and, if desired

70. c) separation of the stereoisomeric mixture;

71. d) building of the RGD cyclic sequence, and if desired

72. e) separation of the stereoisomeric mixture.

73. A process for the stereoselective synthesis of the compounds offormula (I), comprises the following steps:

74. a) Horner-Emmons olefination of a compound of formula (II)

75. wherein

76. R is a lower alkl residue;

77. R₁ is a suitable nitrogen protecting group, to give a compound offormula (III);

78. wherein R₃ is a suitable nitrogen protecting group, R₄ is a loweralkyl residue;

79. b) hydrogenation of said compound of formula (III) by chiralphosphine-Rh catalysed hydrogenation and cyclisation; and, if desired

80. c) separation of the stereoisomeric mixture;

81. d) building of the RGD cyclic sequence and if desired

82. e) separation of the stereoisomeric mixture.

83. Also disclosed are pharmaceutical composition comprising atherapeutically or preventive effective dose of at least a compound offormula (I) in admixture with pharmaceutically acceptable vehiclesand/or excipients.

84. In its broadest aspect, the present invention advantageously teachesa method for selectively inhibiting α_(v)β₃ integrin-mediated cellattachment to an RGD-containing ligand, comprising contacting saidligand with an effective amount of a compound of formula (I), a methodfor treating a subject suffering from altered angiogenesis, comprisingadministering to said subject a compound of formula (I), a method forthe treatment of tumors in a subject comprising administering to saidsubject a compound of formula (I), optionally in combination with otheractive ingredients, in particular other antitumour agents.

85. The present invention shall be described in detail also by means ofexamples and figures, wherein,

BEST MODE FOR CARRYING OUT THE INVENTION

86. The synthesis of so-called peptidomimetics molecules has been a veryactive and productive field of research in drug design (J. Gante, Angew.Chem., Int. Ed. Engl. 1994, 33, 1699. - G. L. Olson, et al.: J. Med.Chem. 1993, 36, 3039. - D. C. Horwell, Bioorg. Med. Chem, Lett. 1993, 3,797. - A. Giannis et al.: Angew. Chem., Int. Ed. Engl. 1993, 32, 1244.B. A. Morgan: Annu. Rep. Med. Chem. 1989, 24, 243). The expectation isthat these molecules will have the same biological effects as naturalpeptides, but at the same time, will be metabolically more stable. Ofparticular interest has been the replacement of reverse-turn dipeptidemotifs with constrained molecules that reproduce their conformationalfeatures (ibid; M. Kahn, Ed., Peptide Secondary Structure Mimetics.Tetrahedron Symposia-in-Print No. 50 1993, 49, 3433-3689 and referencestherein). This goal has been frequently achieved using theazaoxobicyclo[X.Y.O]alkane skeleton and/or heteroatom analogues. Thishas created a demand for efficient synthetic approaches toward suchmolecules, and many methods have been introduced and recently reviewed(S. Hanessian et al: Tetrahedron 1997, 38, 12789-12854). Oneparticularly effective and versatile route has been developed by Lubellet al. and employed for the preparation of enantiopureindolizidinone-type 6,5-fused bicyclic lactams (H.-G. Lombartet al.: J.Org. Chem. 1996, 61, 9437-9446. - F. Polyak et al.: J. Org. Chem. 1998,63, 5937-5949 and references therein for the syntheses ofazabicycloalkane amino acids—F. Gosselin et al.: J. Org. Chem. 1998, 63,7463-7471). Several procedures are also available for the synthesis of7,5-fused bicyclic lactams, the majority of which require relativelylong synthetic sequences. On the contrary, there is not many publishedprotocol that allow the synthesis of 5,5-fused bicyclic lactams.

87. According to the present invention, the beta-turn portion of thecyclic peptide consists in an azabicycloalkane amino acid scaffold,selected from a 5,5-, 6,5- or 7,5-fused bicyclic lactams. Several 6,5-and 7,5-fused 1-aza-2-oxabicyclo[X.3.0]alkane amino acids have beensynthesised, using radical (L. Colombo et al.: Tetrahedron Lett. 1995,36, 625-628. - L. Colombo et al.: Gazz. Chim. It. 1996, 126, 543-554) orionic reactions (L. Colombo et al. Tetrahedron 1998, 54, 5325-5336).These structures can be regarded as conformationally restrictedsubstitutes for Ala-Pro and Phe-Pro dipeptide units, and, if theirconformations meet certain criteria, they can be used to replace thecentral (i+1 and i+2) residues of β-turns.

88. The present invention provides an improved reaction sequence,amenable to large scale preparation, and allowing the synthesis ofdifferent bicyclic lactams from common intermediates, as described inthe appended (FIG. 1).

89. Starting from 5-allyl/formyl prolines 13-18, a Z-selectiveHorner-Emmons olefination followed by double bond reduction has beenused to build the second ring. The starting aldehydes have beenstereoselectively synthesised by modifications of known procedures (videinfra). Stereorandom double bond reduction can be performed using H₂/Pdto yield, after cyclisation, mixtures of easily separable epimers.Stereoselective hydrogenation is studied for the synthesis of 6,5-fusedlactams, and achieved with d.e. 80% using Rh-chiral phosphine catalysts.Structural diversity, in terms of ring size and stereochemistry of theazabicycloalkane fragment, is provided by the new strategy, and accessto the less common 5,5-fused bicyclic scaffold is also secured.

90. Examples of bicyclic dipeptide derivatives 1-12 are shown in FIG. 2.

91. Synthesis of the fused bicyclic lactams 1-12

92. The synthesis of lactams 1-12 follows the common steps reported inFIG. 1. Starting from the cis or trans 5-alkyl proline aldehydes 13-18,a Horner-Emmons olefination with the potassium enolate of(±)-Z-α-phosphonoglycine trimethyl ester (U. Schmidt, A. Lieberknecht,J. Wild, Synthesis 1984, 53-60)sets up the necessary carbon chain.Following protecting group manipulation (vide infra), reduction of theenamino acrylic acids and treatment with condensing agents gives thelactams of both the “cis” and “trans” series in good yields.

93. In all cases where stereoisomeric mixtures of lactams are formed,they can be easily separated by flash chromatography, and theirconfiguration can be assigned with n.O.e, experiments.

94. The synthetic scheme is best illustrated by the synthesis of the6,5-fused “cis”-lactams 2a and 8a (FIG. 3). The necessary cis aldehyde14 is obtained from the known cis 5-allyl-proline derivative 25 (M. V.Chiesa, L. Manzoni, C. Scolastico, Synlett 1996, 441-443) and reactedwith the commercially available phosphonate 26 (U. Schmidt, A.Lieberknecht, J. Wild, Synthesis 1984, 53-60) to give 20 in 98% yieldand 7:1 Z:E ratio.

95. Hydrogenation of 20 occurs initially at the enamino Cbz group, andthus results in a complex mixture of products. To circumvent thisproblem, the substrate is treated with Boc₂O to give 27 (98%). Reductionof 27 with H₂/Pd(OH)₂ followed by reflux in MeOH gives a 1:1 mixture of8a and 2a, which are easily separated by flash-chromatography. From 14the whole sequence requires only two chromatographic separations(purification of 20 and separation of 8a from 2a) and can easily becarried out in multigram scale.

96. The stereoselective preparation of the two epimers 8a and 2a (FIG.3) is carried out using chiral phosphine-Rh catalysed hydrogenation ofthe enamino acid 28.

97. Chiral phosphine-Rh catalyst is well-known to represent a powerfuland well-established way of access to naturally and non-naturallyoccurring amino acids and the catalytic asymmetric hydrogenation ofdehydropeptides is the logical extension of this methodology to thepreparation of biologically active chiral oligo- and polypeptides.

98. In asymmetric catalytic hydrogenations using chiral phosphine-Rhcatalysts (Z) olefins usually gives the highest stereoisomeric purity ofthe products, but the most stringent requirement for the substrateremains the presence of an acetamido or an equivalent group on thedouble bond. (K. E. Koenig in Asymmetric Synthesis, J. D. MorrisonEditor, Vol 5, Academic Press Inc. 1985, 71) The amide-type carbonyl isneeded in order to allow two-point co-ordination of the substrate to themetal, which increases the sterical demand as it has been fullyelucidated experimentally.(J. Halpern, ibidem, 41) For applications tothe synthesis of peptides protecting groups other than the acetamido,like Boc or Cbz should be used, thus permitting differentialdeprotection. However, very few examples of asymmetric catalytichydrogenation are known in which these protecting groups are found onthe enamino nitrogen: (B. Basu, S. K. Chattopadhyay, A. Ritzen, T.Frejd, Tetrahedron Asymmetry, 1997, 8, 1841) (S. D. Debenham, J. D.Debenham, M. J. Burk, E. J. Toone, J. Am. Chem. Soc. 1997, 119, 9897)more frequently Boc or Cbz protecting groups are present in differentposition of dehydropeptides being hydrogenated at the N-terminus. (A.Hammadi et al. Tetrahedron Lett. 1998, 39, 2955 - I. Ojima, Pure & Appl.Chem, 1984, 56, 99). For the catalytic asymmetric hydrogenation of 28[Rh(Phosphine)(COD)]ClO₄ catalysts is used. The catalysts were preparedby displacing one cyclooctadiene ligand of [Rh(COD)₂]ClO₄ with theappropriate phosphine. The ligands investigated are (R)-Prophos 29 and(+) or (−) BitianP 30 and 31. BitianP is a chiral atropisomericchelating phosphine belonging to a new class of ligands based onbiheteroaromatic framework, which gives very high e.e. % in theasymmetric hydrogenation of olefins and ketones. (E. Cesarotti et al. J.Chem. Soc. Chem. Comm. 1995, 685 - Cesarotti et al. J. Org. Chem. 1996,61, 6244).

99. The results of asymmetric hydrogenation are reported in the Table 1.The conversion is always quantitative but the higheststereodifferentiation is obtained with [Rh/(−)-BitianP](entry 3). Theresults suggest that the newly created stereocentre is mainly determinedby the catalyst, which overruns the effect of the stereocentre on thesubstrates (entry 2 and 3). The results also indicate that the Bocprotecting group on the enamino nitrogen fulfils the requirements andallows the olefin to chelate to the catalyst. TABLE 1 Asymmetrichydrogenation of 28 Entry Catalyst 32/33 d.e. % 1 Rh-29 86/14 72 2 Rh-3013/87 74 3 Rh-31 90/10 80

100. Reactions were carried out at R.T. for 24 h under 10 atm of H₂

101. Treatment of crude 32 and 33 with CH₂N₂, followed by hydrogenationand cyclisation under the usual conditions (H₂/Pd-C followed by refluxin MeOH) allows a stereoselective route to lactams 8a and 2a.

102. All the remaining lactams 1-12 can be synthesised followingessentially the same sequence described above. Thus, the 7,5-fusedlactams 3a and 9a (FIG. 4) can be made starting from the cis aldehyde15, easily prepared from the cis 5-allyl proline 25.(M. V. Chiesa, L.Manzoni, C. Scolastico, Synlett 1996, 441-443) Horner-Emmons reaction of15 with 26 gives a 6:1 Z:E mixture of enamino acrylates. AfterN-protection they are reduced with H₂/Pd-C. The thermic cyclisation ofmethyl ester 34 can be carried out n a suitable solvent, for examplexylene. Better results are obtained upon ester hydrolysis followed byEDC/HOBT promoted lactam formation to give 3a and 9a, which are easilyseparable by flash chromatography (51% overall yield from 25).

103. The starting material for the synthesis of the 5,5-fused “cis”lactams (FIG. 5) is alcohol 36. Oxdation and Horner-Emmons is reactionwith 26 followed by N-Boc protection gives 37 as a 5:1 Z:E mixture in57% yield. Hydrogenation of 37 (H₂/Pd(OH)₂) results in a complex mixtureof products, from which the 1,2 diamino ester 38 is anyway isolated in40% yield. The formation of 38 may result from initial N-debenzylationof 37 followed by intramolecular Michael addition to the enamino esterdouble bond and hydrogenolysis of the resulting aziridine. The problemcan be partly circumvented by performing the hydrogenation starting fromthe acid 39. Treatment of 39 with H₂/Pd-C followed by reflux in MeOHgives an easily separable 1:1 mixture of 1a and 7a in 40% yield.

104. An alternative synthesis of these lactams is also provided startingfrom the trifluoroacetamido aldehyde 13 (FIG. 6). Aldehyde 13 issynthesised from 36 with a series of 5 high-yielding steps.Horner-Emmons and nitrogen protection gives 40 (46% over 7 steps), whichcould be directly reduced to give a 1:1 mixture of the fully protectedester 41 (77%). Removal of the trifluoroacetamido protecting group(NaBH₄ in MeOH, 84%) followed by treatment in refluxing xylene gives thelactams 1a and 7a in 78% yield.

105. The same synthetic schemes are equally adopted for the synthesis ofthe “trans” lactam series.

106. Starting material for the 6,5-fused “trans” lactams 5a and 11a isthe trans-substituted proline 17 (FIG. 7). Aldehyde 17 is best obtainedfrom ester 43, which is made in one step from N-Cbz-5-hydroxy prolinetert-Butyl ester as 4:1 trans:cis mixture, following a publishedprocedure. (I. Collado et al., Tetrahedron Lett., 1994, 43, 8037) TheHorner-Emmons reaction with the potassium enolate of 26 proceeds with98% yield. Treatment with Boc₂O and cis/trans isomers separation,followed by unselective H₂/Pd-C hydrogenation of the crude and treatmentin refluxing MeOH gives a 1:1 mixture of easily separated 5a and 11a.

107. Finally, synthesis of the 7,5-fused “trans” lactams 6a and 12a isachieved starting from the “trans” allyl proline 45 (FIG. 8). (M. V.Chiesa et al. Synlett 1996, 441-443) Hydroboration and Swern oxidation(80% over 2 steps) gives the aldehyde 18, which reacted with 26 to give,after nitrogen protection, 46 as a 6:1 Z:E mixture. The usual sequence(NaOH; H₂/Pd-C) allowed the isolation of 6a and 12a in 40% overallyield.

108. As far as the synthesis of the cyclic RGD portion, syntheticmethods are well known in the art. It is convenient to use the solidphase synthesis approach, although other methods could be used.

109. The classical solid-phase synthesis is preferred.

110. The solid-phase synthesis is carried out as outlined in C. Gennariet al. Eur. J. Org. Chem. 1999, 379-388.

111. The protected amino acid is condensed on a suitable resin, forexample a Wang-Merrifield resin. Protecting groups are known in thisart. 9-fluorenylmethoxycarbonyl (FMOC) is preferred

112. After having activated the resin, N-FMOC-Gly is attached to theWang-Merrifield resin by means of a suitable condensing agent,preferably diisopropylcarbodiimide (DIC)/1-hydroxybenzotiazole(HOBt)/4-dimethylaminopyridine (DMAP) (J. Org, Chem, 1996, 61,6735-6738.

113. Subsequently, N-FMOC-Arg(Pmc)OH is attached, followed by thebicyclic N-FMOC-lactam (IIIa) or (IIIb) and finally N-FMOC-Asp(tBu)OH.

114. The compounds of the present invention are endowed with interestingphysiological properties, which make them useful as medicaments. Inparticular, the compounds of formula (I) herein disclosed are selectiveantagonists of α_(v)β₃ integrins. This antagonist activity provides theuse of said compounds for the preparation of medicaments useful ininhibiting the action of α_(v)β₃ integrins. In particular, saidmedicaments will be used in the treatment of tumors, namely ininhibiting tumor growth and/or angiogenesis or metastasis.

115. As far as the industrial aspects of the present invention areconcerned, the compounds of formula (I) shall be suitably formulated inpharmaceutical compositions. Said compositions will comprise at leastone compound of formula (I) in admixture with pharmaceuticallyacceptable vehicles and/or excipients. According to the therapeuticnecessity, the bioavailability of the selected compound, itsphysico-chemical characteristics, the pharmaceutical compositionsaccording to the present invention will be administered by enteral orparenteral route. Enteral pharmaceutical compositions may be both in theliquid or solid from, for example tablets, capsules, pills, powders,sachets, freeze dried powders to be readily dissolved or in any otherway soluble powders, solutions, suspensions, emulsions. Parenteralformulation will be in injectable form, as solutions, suspensions,emulsions or in powdery form to be dissolved immediately before use.Other administration routes are also provided, for example intranasal,transdermal or subcutaneous implant. Special pharmaceutical compositionscan also be provided. For example controlled release formulations orparticular vehicles, for example liposomes.

116. The preparation of the pharmaceutical compositions according to thepresent invention is absolutely within the general knowledge of theperson skilled in this art.

117. The dosage will be established according to the type of thepathology to be treated, its severity, and the conditions of the patient(weight, age, and sex).

118. The following examples further illustrate the invention.

119. General: ¹H and ¹³C NMR spectra were recorded in CDCl₃ or C₆D₆ asindicated, at 200 (or 300) and 50.3 MHz, respectively. The chemicalshift values are given in ppm and the coupling constants in Hz. Opticalrotation data were obtained on Perkin-Elmer model 241 polarimeter.Thin-layer chromatography (TLC) is carried out using Merck precoatedsilica gel F-254 plates. Flash chromatography is carried out with MerckSilica Gel 60, 200-400 mesh. Solvents were dried with standardprocedure, and reactions requiring anhydrous conditions were performedunder a nitrogen atmosphere. Final product solutions were dried overNa₂SO₄, filtered and evaporated under reduced pressure on a Buchi rotaryevaporator.

EXAMPLE 1 Preparation of enamides via Horner-Emmons reaction

120. General procedure A: To a stirred solution of tBuOK (7.36 mmol) in40 ml of dry CH₂Cl₂ under nitrogen atmosphere, at −78° C., was added asolution of Z-α-phosphonoglycine trimethyl ester 26 (7.36 mmol) in 5.0ml of dry CH₂Cl₂. The solution was stirred for 30 min at thistemperature and then a solution of aldehyde (6.13 mmol) in dry CH₂Cl₂(25 ml) was added. After 5 hours the solution was neutralised with aphosphate buffer. The aqueous phase was extracted with CH₂Cl₂, driedover Na₂SO₄ and the solvent evaporated under reduced pressure. The crudewas purified by flash chromatography (hexane/ethyl acetate), affordingthe enamide in a Z:E diastereoisomeric mixture.

Preparation of N-Boc-protected enamide

121. General procedure B: A solution of encode (11.0 mmol), (Boc)₂O(22.0 mmol) and a catalytic quantity of DMAP in 40 ml of dry THF, wasstirred for 30 min. under nitrogen. The solution was then quenched with40 ml of water and extracted with ethyl acetate. The organic phase wasdried over Na₂SO₄ and the solvent evaporated under reduced pressure. Thecrude was purified by flash chromatography (hexane/ethyl acetate),yielding the Boc-protected enamide.

Preparation of alcohol via hydroboration

122. General procedure C: To a solution of allyl proline (2.34 s mmol)in dry THF (4.2 ml) was added a 0.5 M solution of 9-BBN in THF (1.26mmol). The reaction was stirred for 12 h. and then cooled at 0° C. and,water (0.6 ml), a 3 N solution of NaOH (0.5 ml) and H₂O₂ 30% (0.44 ml)were added. The reaction was stirred for 1 h. at room temperature andthen refluxed for other 2 h. The aqueous phase was extracted with AcOEt,the collected organic phases were dried over Na₂SO₄, filtered andevaporated under reduced pressure, the crude was purified by flashchromatography (hexane/ethyl acetate), yielding the alcohol as yellowoil.

Preparation of aldehyde via Swern oxidation

123. General procedure D: To a stirred solution of oxalyl chloride (16.9mmol) in 35 ml of CH₂Cl₂, cooled at −60° C., were added DMSO (23.1mmol), alcohol (5.66 mmol) dissolved in 21 ml of CH₂Cl₂, TEA (28.2mmol). The reaction was warmed at room temperature. After one hour thereaction was washed with 50 ml of water and the aqueous phase wasextracted with CH₂Cl₂. The collected organic layers were dried overNa₂SO₄. The solvent was evaporated under reduced pressure and the crudepurified by flash chromatography (hexane/ethyl acetate), yielding thealdehyde.

EXAMPLE 2 Aldehyde (14)

124. A stirred solution of 25 (6.0 g, 17.4 mmol) in 84 ml of CH₂Cl₂ wascooled at −60° C. and bubbled with O₃ (flow rate=30 l/hour) After 1.5hours the reaction was allowed to warm to room temperature and bubbledwith N₂ in order to eliminate the excess of O₃. The solution was thencooled at 0° C. with an ice bath and Me₂S (101.8 mmol, 38 ml) was added.After 5 days of stirring at room temperature the solvent was evaporatedunder reduced pressure and the crude was purified by flashchromatography (hexane/ethyl acetate, 8:2), yielding 4.53 g of 14 (75%)as yellow oil. - [α]_(D) ²²=−22.03 (c=1.27, CHCl₃), −¹H NMR (200 MHz,CDCl₃), (signals were splitted for amidic isomerism): δ=1.4-1.5 [2 s, 9H, C(CH₃)₃], 1.6-2.4 (m, 4 H, CH₂—CH₂), 2.4-3.2 (2 m, 2 H, CH₂CHO),4.3-4.5 (m, 2 H, CH₂—CH—N, N—CH—COOtBu), 5.15 (s, 2 H, CH₂Ph), 7.30 (m,5 H, aromatic), 9.8 (2 s, 1 H, CHO). - ¹³C NMR (50.3 MHz, CDCl₃)(signals were splitted for amidic isomerism): δ=200.8, 171.7, 154.0,136.2, 128.3, 128.0, 127.8, 127.6, 81.4, 67.0, 66.9, 60.8, 60.3, 54.0,53.2, 49.0, 48.3, 31.0, 30.2, 29.5, 28.9, 28.0, 27.7. - FAB⁺MS: calcd.for C₁₀H₂₅NO₅ 347.4, found 348.

EXAMPLE 3 Enamide (20)

125. The general procedure A was followed using 14 and the crude waspurified by flash chromatography (hexane/ethyl acetate, 65:35),affording 20 (98%) in a 7:1 Z:E ratio as colourless oils. Z-isomer: -[α]_(D) ²²=30 38.78 (c=1.26, CHCl₃), ¹H NMR (200 MHz, CDCl₃) (signalswere splitted for amidic isomerism): δ=1.3-1.5 [2 s, 9 H, C(CH₃)₃],1.5-2.3 (m, 4 H, CH₂—CH₂), 2.4-2.7 (2 m, 2 H, ═CH—CH₂), 3.7 (2 s, 3 H,COOCH₃), 4.2 (2 m, 2 H, —CH₂—CH—N, N—CH—COOtBu), 5.10 (m, 4 H, CH₂Ph),6.15 (m, 1 H, CR₂═CH), 7.30 (m, 10 H, aromatic).- ¹³C NMR (50.3 MHz,CDCl₃) (signals were splitted for amidic isomerism): δ=172.4, 164.9,154.5, 136.2, 132.5, 128.3, 128.2, 127.8, 127.7, 127.6, 81.8, 67.2,66.9, 60.8, 60.3, 57.9, 57.2, 52.1, 33.8, 33.2, 30.7, 29.8, 29.5, 29.0,28.0, 27.7, 27.6. - FAB⁺MS: calcd. for C₃₀H₃₆N₂O₈ 552.6, found 553. -E-isomer: - [α]_(D) ²²=−4.08 (c=1.17, CHCl₃). ¹H NMR (200 MHz, CDCl₃)(signals were splitted for amidic isomerism): δ=1.25-1.50[3 s, 9 H,C(CH₃)₃], 1.5-2.3 (m, 4 H, CH₃—CH₃), 2.8-3.3 (2 m, 2 H, ═CH—CH₂), 3.8 (2s, 3 H, COOCH₃), 4.1 (m, 1 H, —CH₂—CH—N), 4.25 (m, 1 H, N—CH—COOtBu),5.15 (2 s, 4 H, CH₂Ph), 6.30 (m, 1 H, ═CH), 7.30 (m, 10 H, aromatic). -¹³C NMR (50.3 MHz, CDCl₃) (signals were splitted for amidic isomerism):δ=171.8, 164.4, 154.1, 153.6, 136.4, 135.9, 128.7, 128.4, 128.2, 128.1,128.0, 127.8, 127.7, 127.6, 126.5, 125.9, 81.2, 80.9, 66.7, 61.0, 60.6,60.2, 58.8, 58.1, 52.2, 32.7, 32.0, 31.8, 29.9, 29.5, 29.2, 28.8, 27.8,27.7, 22.5, 14.0.

EXAMPLE 4 Enamide (27)

126. The general procedure B was followed using 20 and the resultingcrude was purified by flash chromatography (hexane/ethyl acetate, 7:3),yielding 27 (98%) as yellow oil. - Z-isomer. - [α]_(D) ²²=+16.95(c=1.86, CHCl₃). - ¹H NMR (200 MHz, CDCl₃) (signals were splitted foramidic isomerism): δ1.3-1.5 [2 s, 18 H, C(CH₃)₃], 1.6-2.2 (m, 4 H,CH₂—CH₂), 2.3-2.8 (2 m, 2 H, ═CH—CH₂), 3.7 (s, 3 H, COOCH₃), 4.1-4.2 (2m, 2 H, ═CH—CH₂—CH—N, N—CH—COOtBu), 5.15 (m, 4 H, CH₂Ph), 6.95 (dd,J=8.5, J=6.4 Hz, 1 H, ═CH), 7.30 (m, 10 H, aromatic). - ¹³C NMR (50.3MHz, CDCl₃) (signals were splitted for amidic isomerism): δ=171.4,163.8, 154.6, 154.3, 152.1, 150.4, 139.0, 138.8, 136.2, 135.1, 129.7,128.3, 128.2, 128.1, 127.8, 127.6, 83.3, 81.2, 77.1, 68.2, 66.8, 60.9,60.4, 57.5, 56.7, 52.1, 32.8, 32.1, 29.9, 29.1, 28.8, 27.7. -E-isomer: - [α]_(D) ²²=+7.34 (c=1.33, CHCl₃). -¹H NMR (200 MHz, CDCl₃)(signals were splitted for amidic isomerism): δ=1.3-1.5 [2s, 18 H,C(CH₃)₃], 1.6-2.2 (m, 4 H, CH₂—CH₂), 3.0-3.3 (m, 2 H, —CH—CH₂), 3.75 (2s, 3 H, COOCH₃), 4.1-4.2 (2 m, 2 H, ═CH—CH₂—CH—N, N—CH—COCR), 5.1-5.2(m, 4 H, CH₂Ph), 6.3 (m, 1 H, ═CH), 7.30 (m, 10 H, aromatic). - ¹³C NMR(50.3 MHz, CDCl₃) (signals were splitted for amidic isomerism): δ=171.6,163.8, 154.5, 154.3, 152.1, 150.4, 142.8, 142.5, 136.3, 135.2, 128.7,128.3, 128.2, 128.1, 127.9, 127.8, 127.6, 83.2, 81.1, 68.2, 66.8, 61.1,60.6, 58.1, 57.4, 51.7, 32.7, 32.0, 29.5, 29.4, 28.9, 28.7, 27.7.

EXAMPLE 5 6,5-Fused bicyclic lactam (2a, 8a)

127. A solution of 0.320 g of 27 (0.49 mmol) and a catalytic quantity ofPd/C 10% in 5 ml of MeOH was stirred under H₂ for one night. Thecatalyst was then filtered through celite and the filtration bed waswashed with MeOH. The solvent was evaporated under reduced pressure, theresidue was dissolved in MeOH and refluxed for 48 h. The solvent wasremoved and the two diastereoisomers formed were separated by flashchromatography (hexane/ethyl acetate, 7:3), yielding 0.122 g of 8a and2a (70%) in a 1.4:1 diastereoisomeric ratio as white foam. - [α _(D)²²=−10.70 (c=1.29, CHCl₃). - ¹H NMR (200 MHz, CDCl₃): δ=1.43-1.45 [2 s,18 H, C(CH₃)₃], 1.5-2.5 (m, 8 H, CH₂—CH₂, BocN—CH—CH₂—CH₂), 3.69 [m, 1H, CH—N], 4.1 (m, 1 H, CH—NBoc), 4.38 (dd, J=7.7 Hz, J=1.8 Hz, 1 H,N—CH—COOtBu), 5.59 (d, J=5.4 Hz, 1 H, NH). - ¹³C NMR (50.3 MHz, CDCl₃):δ=170.7, 165.8, 155.8, 147.1, 81.4, 79.3, 59.0, 56.2, 49.9, 32.0, 29.5,29.1, 28.2, 27.8, 27.0, 26.5. - FAB⁺MS: calcd. for C₁₈H₃₂N₂O₅ 354.46,found 354. - 8a [α]_(D) ²²=−45.07 (c=1.69, CHCl₃), - 1H NMR (200 MHz,CDCl₃): δ=1.44-1.46 [2 s, 18 H, C(CH₃)₃], 1.55-2.2 (m, 7H, CH₂—CH₂,BocN—CH—CHH—CH₂), 2.5 (m, 1H, BocN—CH—CHH), 3.75 !tt, J=11.2 Hz, J=4.2Hz, 1 H, CH—N], 3.90 (m, 1 H, CH—NBoc), 4.32 (d, J=9.2 Hz, 1 H,N—CH—COOtBu), 5.59 (broad, 1 H, NH). - 13C NMR (50.3 MHz, CDCl₃):δ=170.6, 167.9, 155.7, 81.2, 79.4, 77.5, 60.4, 59.0, 52.2, 31.4, 28.5,28.3, 28.2, 27.8, 27.6. - FAB⁺MS: calcd. for C₁₈H₃₂N₁O₅ 354.46, found354.

Acid (28)

128. To a solution of 27 (0.640 g, 0.980 mmol) in 4.9 ml of MeOH wasadded 4.9 ml of 1N NaOH (4.9 mmol). After 18 hours of stirring at roomtemperature the solvent was evaporated under reduced pressure. The solidresidue was dissolved in 5 ml of water and 2N HCl was added until pH 3,then the aqueous solution was extracted with CH₂Cl₂. The organic phasewas dried with Na₂SO₄, the solvent evaporated under reduced pressure andthe crude was purified by flash chromatography (CH₂Cl₂/MeOH, 95:5),yielding 0.420 g of 28 (85%) as a white solid.

129. Z isomer: - [α]_(D) ²²=−57.01 (c=1.99, CHCl₃). - ¹H NMR (200 MHz,CDCl₃) (signals were splitted for amidic isomerism): δ=1.30-1.50 [2 s,18 H, C(CH₃)₃], 1.7-2.7 (m, 6 H, CH₂—CH₂, ═CH—CH₂), 4.2-4.3 (m, 2 H,═CH—CH₂—CH—N, N—CH—COOtBu), 5.1 (m, 2 H, CH₂Ph), 6.6 (m, 1 H, ═CH), 7.30(m, 6 H, aromatic, NHBoc). - ¹³C NMR (50.3 MHz, CDCl₃) (signals weresplitted for amidic isomerism): δ=171.5, 168.3, 154.8, 154.5, 140.6,136.4, 136.1, 133.9, 133.5, 128.3, 128.2, 128.1, 127.8, 127.4, 126.9,81.3, 80.9, 67.1, 66.9, 65.0, 66.9, 65.0, 57.5, 56.8, 33.4, 32.4, 29.5,28.5, 28.5, 28.0, 27.8, 27.7, 27.4.

130. E isomer: - [α]_(D) ²²=−41.63 (c=1.87, CHCl₃). - ¹H NMR (200 MHz,CDCl₃) (signals were splitted for amidic isomerism): δ=1.35-1.50[3 s, 18H, C(CH₃)₃], 1.7-2.4 (m, 4 H, CH₂—CH₂), 2.7-3.2 (m, 2 H, ═CH—CH₂),4.2-4.3 (m, 2 H, ═CH—CH₂—CH—N, N—CH—COOtBu), 5.1 (m, 2 H, CH₂Ph),6.7-6.9 (m, 2 H, ═CH, NHBoc), 7.30 (m, 5 H, aromatic). -¹³C NMR (50.3MHz, CDl₃) (signals were splitted for amidic isomerism): δ=171.7, 167.2,154.9, 154.5, 154.3, 136.5, 136.2, 128.3, 128.2, 127.7, 127.5, 126.9,126.3, 126.1, 81.2, 80.4, 66.9, 65.0, 60.7, 60.4, 58.3, 57.7, 32.9,32.0, 29.5, 28.4, 28.1, 27.8, 27.7, 27.4, 27.1, 14.0.

Acid (32, 33)

131. To the [Rh-(−)-BitianP] catalyst prepared as described in theliterature was added 28 (0.16 mmol) and MeOH (30 ml), the resultingsolution was stirred for 30 min. A 200 ml stainless-steel autoclaveequipped with a magnetic stirrer and a thermostatic bath waspressuarised with hydrogen and vented three times. The solution wastransferred into the autoclave with a syringe and the autoclave waspressurised at 10 KPa with hydrogen. The solution was stirred for 24 h.at 30 ° C. The hydrogen pressure was released, the solvent evaporated.The crude was submitted to the next reaction without furtherpurification.

6,5-fused bicyclic lactam (2a)

132. To a solution of 32 and 33 as diastereomeric mixture in MeOH (1.5ml) was added a solution of CH₂N₂ in Et₂O until the TLC showed that thereaction was complete. The solution was evaporated and the crude wasdissolved in MeOH (2 ml) and a catalytic quantity of Pd/C was added, themixture was stirred under H₂ for 12 h. The catalyst was then filteredthrough celite pad and washed with MeOH. The solvent was evaporatedunder reduced pressure and the crude, as a white foam, was refluxed inMeOH for 48 h. The solvent was evaporated under reduced is pressure andthe crude was purified by flash chromatography (hexane/ethyl acetate7:3) affording 2a (85%) as a white solid.

EXAMPLE 6 6,5-fused bicyclic lactam (8a)

133. This bicyclic lactam was achieved with the same synthetic sequencefollowed for the lactam 2a using for the asymmetric hydrogenation the[Rh-(+)-BitianP] catalyst.

Aldehyde (15)

134. The general procedure C was followed using 25 and the resultingresidue was purified by flash chromatography (hexane/ethyl acetate,7:3), yielding the alcohol (95%) as yellow oil. - ¹H NMR (200 MHz,CDCl₃) δ=1.4 [s, 9 H, C(CH₃)₃], 1.6-2.4 (m, 8 H, CH₂—CH₂), 3.5-3.8 (2 m,2 H, CH₂OH), 4.1 (m, 1 H, CH₂—CH—N), 4.25 (m, 1 H, N—CH—COOtBu), 5.15(s, 2 H, CH₂Ph), 7.30 (m, 5 H, aromatic).

135. The general procedure D was followed using the previous alcohol andthe resulting crude residue was purified by flash chromatography(hexane/ethyl acetate, 7;3), yielding 15 (89%) as an oil. - ¹H NMR (200MHz, CDCl₃), (signals were splitted for amidic isomerism): δ=1.4-1.5 [2s, 9 H, C(CH₃)₃], 1.6-2.8 (m, 4 H. CH₂—CH₂), 4.05 (m, 1 H, CH₂—CH—N),4.25 (m, 1 H, N—CH—COOtBu), 5.15 (s, 2 H, CH₂Ph), 7.30 (m, 5 H,aromatic), 9.6-9.8 (2 s, 1 H, CHO).

Aminoester (34)

136. The general procedure A was followed using 15 and the resultingresidue was purified by flash chromatography yielding the enamide (95%)as yellow oil. The compound previously synthesised was submitted to thegeneral procedure B and the resulting residue was purified by flashchromatography yielding the N-Boc protected compound (95%) as whitesolid. A solution of this compound (0.96 mmol) in MeOH (1 mL) and acatalytic quantity of Pd/C were stirred under hydrogen atmosphere for 12h. The catalyst was then filtered through a celite pad. The solvent wasevaporated under reduced pressure yielding 0.320 g of 34 (83%) as awhite solid (mixture of two diastereoisomers). - ¹H NMR (200 MHz,CDCl₃): δ=1.47, 1.48 [2 s, 18 H, C(CH₃)₃], 1.40-2.1 (m, 10 H, CH₂—CH₂,BocN—CH—CHH—CH₂), 3.00 (m, 1 H, CH—N), 3.6 (m, 1 H, N—CH—COOtBu), 4.3(m, 1 H, CH—NBoc), 5.05 (db, 1H, NH).

Amino acid (35)

137. To a solution of 34 (0.288 g, 0.720 mmol) in MeOH was added 1NNaOH, after 1.5 h. the solution was acidified until pH 3 with 1N HCl,then the solution was evaporated. The crude was submitted to the nextreaction without further purification.

EXAMPLE 7 7,5-fused bicyclic lactams (3a, 9a)

138. To a solution of the crude 35 (0.720 mmol) in CH₂Cl₂ (80 ml) wasadded in the order: Et₃N (0.720 mmol, 0.220 ml), HOBt (0.166 g, 1.22mmol) and a catalytic quantity of DMAP. After 15 min was added EDC(0.180 g, 0.937 mmol) and the solution was stirred for 24 h. To thesolution was added H₂O (40 ml), the aqueous phase was extracted withCH₂Cl₂ and the collected organic layers were dried with Na₂SO₄ filteredand evaporated under reduced pressure affording 0.191 g of 3a and 9a ina 1:1 diastereoisomeric ratio and 72% of yield over 2 steps.

139. (3a). ¹H NMR (200 MHz, CDCl₃): δ=1.41, 1.42 [2 s, 18 H, C(CH)₃],1.5-2.5 (m, 10 H, CH₂—CH₂), 3.80 (m, 1 E, CH—N), 4.2 m, 1 H, CH—NBoc),4.51 (dd, J=4.8 Hz, 1H, N—CH—COOtBu), 5.54 (db, 1 H, NH). - (9a). - ¹HNMR (200 MHz, CDCl₃): δ=1.42, 1.43 [2 S, 18 H, C(CH₃)₃], 1.50-2.2 (m,10H, CH₂—CH₂), 3.8 [m, 1 H, CH—N], 4.25 (dd, J=4.6 Hz, J=9.6 Hz, 1 H,CH—NBoc), 4.42 (dd, J=2.3 Hz, J=7.2 Hz, 1 H, N—CH—COOtBu), 5.30 (bs, 1H, NH).

140. Enamide (37): The general procedure D was followed using 36 and thecrude was purified by flash chromatography (hexane/ethyl acetate, 7:3),yielding the aldehyde (81%) as an oil. - ¹H NMR (200 MHz, CDCl₃),(signals were splitted for amidic as isomerism): δ=1.48[s, 9 H,C(CH₃)_(3b ], 1.8)-2.2 (m, 4 H, CH₂—CH₂), 3.21 (m, 1 H, CH₂—CH—N), 3.45(m, 1 H, N—CH—COOtBu), 3.70 (d, J=12 Hz, 1 H, HCHPh), 4.10 (d, J=12 Hz,1 H, HCHPh), 7.30 (m, 5 H, aromatic), 9.12 (d, 1 H, CHO).

141. The general procedure A was followed using the previous aldehydeand the crude was purified by flash chromatography (hexane/ethylacetate, 65:35), affording the enamide (98%) in a 9:1 Z:E ratio ascolourless oils. Z-isomer - ¹H NMR (200 MHz, CDCl₃) δ=1.31 [s, 9 H,C(CH₃)₃], 1.7-2.2 (m, 4 H, CH₂—CH₂), 3.3 (m, 1 H, N—CH—COOtBu) 3.5 (s, 1H. CH₂—CH—N), 3.66 (d, J=13.2 Hz, HCHPh) 3.73 (s, 1 H, COOCH₃), 3.79 (d1 H, HCHPh), 5.11 (d, J=12.5 Hz, 1 H, OHCHPh), 5.15 (d, J=12.5 Hz, 1 H,OHCHPh), 6.07 (d, J=7.4 Hz, 1 H, ═CH), 7.10-7.6 (m, 10 H, aromatic),8.15 (sb, 1 H, —NH). - ¹³C NMR (50.3 MHz, CDCl₃): δ=173.7, 165.1, 154.1,137.4, 136.1, 129.5, 128.5, 128.3, 128.0, 127.8, 127.7, 127.1, 80.5,66.9, 65.3, 62.3, 57.5, 52.0, 30.1, 28.9, 27.7.

142. The general procedure B was followed using the enamide previoussynthesised. The crude was purified by flash chromatography(hexane/ethyl acetate, 7:3) yielding 37 (98%) as a white solid. - ¹H NMR(200 MHz, CDCl₃) (signals were splitted for amidic isomerism): δ=1.3-1.5[2 s, 18 H, C(CH₃)₃], 1.6-2.2 (m, 4 H, CH₂—CH₂), 3.1 (m, 1 H,N—CH—COOtBu), 3.5 (m, 1 H, CH₂—CH—N), 3.7 (s, 1 H, COOCH₃), 3.7 (d, J=12Hz, 1 H, HCHPh), 3.9 (d, J=12 Hz, 1 H, HCHPh), 5.20 (d, J=12 Hz, 1 H,HCHPh), 7.0 (d, J=8.6 Hz, 1 H, ═CH), 7.1-7.4 (m, 10 H, aromatic).

143. Amino acid (39): To a solution of 37 (0.424 g, 0.713 mrnol) in MeOH(4 ml) was added IN NaOH (4 mmol, 4 ml) and stirred for 1.5 h. Thesolution was acidified until pH 3 with 1N HCl, then the 20 solution wasevaporated. The crude was submitted to the next reaction without furtherpurification. - ¹H NMR (200 MHz, CDCl₃) (signals were splitted foramidic isomerism): δ=1.35, 1.5 [2 s, 18 H, C(CH₃)₃], 1.7-2.3 (m, 4 H,CH₂—CH₂), 3.3 (m, 1 H, N—CH—COOtBu), 3.65 (m, 1 H, CH₂—CH—N), 3.7 (d,J=12.8 Hz, 1 H, HCHPh), 3.9 (d, J=12.8 Hz, 1 H, HCHPh), 6.5 (d, J=7.6Hz, 1 H. ═CH), 7.1-7.4 (m, 10 H, aromatic), 9.00 (bs, 1 H, —COOH).

EXAMPLE 8 5,5-fused bicyclic lactams (1a, 7a)

144. A solution of 39 (0.713 mmol) and a catalytic quantity of Pd(OH)₂/C20% in 1 ml of MeOH (7 ml) was stirred under hydrogen atmosphere for12h. The catalyst was then filtered through a celite pad and the solventwas evaporated under to reduced procedure. The crude was dissolved inMeOH and refluxed for 48 h. The solvent was evaporated under reducedpressure and the crude was purified by flash chromatography(hexane/ethyl acetate 6:4) affording 0.097 g of 1a and 7a as a whitesolid in 40% of yield (over 2 steps) and 1:1 diastereomeric ratio. 1a. -[α]_(D) ²²=−4.80 (c=1.20, CHCl₃). - ¹H NMR (200 MHz, CDCl₃): δ=1.50,1.51 [2 s, 18 H, C(CH₃)₃], 1.6-2.4 (m, 5 H, CH₂—CH₂, BocN—CH—CHH), 2.95(m, 1 H, BocN—CH—CHH), 3.85 [m, 1 H, (CH—N], 4.15 (d, J=8.8 Hz, 1 H,N—CH—COOtBu), 4.60 (m 1 H, CH—NBoc), 5.25 (broad, 1 H, NH). - ¹³C NMR(50.3 MHz, CDCl₃) (signals were splitted for amidic isomerism): δ=171.7,169.7, 155.6, 81.8, 79.5, 58.8, 56.5, 56.0, 55.8, 39.5, 33.4, 29.5,28.2, 27.8. - FAB⁺MS: calcd. for C₁₇H₂₈N₂O₅ 340.41, found 341. - 2a:[α]_(D) ²²=−4.80 (c=1.20, CHCl₃). - ¹H NMR (200 MHz, CDCl₃): δ=1.45 [2s, 18 H, C(CH₃)₃], 1.5-2.5 (m, 6 H, CH₂—CH₂, BocN—CH—CH₂), 4.05 (d,J=8.8 Hz, 1 H, N—CH—COOtBu), 4.12 (m, 1 H, CH—N), 4.25 (m, 1 H,CH—NBoc), 5.05 (broad, 1 H, NH).- ¹³C NMR (50.3 MHz, CDCl₃) (signalswere splitted for adic isomerism); δ=170.9, 169.8, 155.2, 82.2, 81.8,79.9, 77.1, 61.2, 58.8, 57.6, 56.0, 55.8, 34.4, 33.8, 33.4, 29.9, 29.5,29.2, 28.5, 28.1, 27.7. - FAB⁺MS: calcd. for C₁₇H₂₈N₂O₅ 340.41, found341.

Aldehyde (13)

145. To a stirred solution of 36 (1.5 g, 5.14 mmol) in 39 ml of to dryCH₂Cl₂ under nitrogen were added in the order: TBDMSCl (0.931 g, 6.17mmol), TEA (6.17 mmol, 0.94 ml) and DMAP (0.063 g, 0.51 mmol). After 12h. the solvent was evaporated under reduced pressure and the crudepurified by flash chromatography (hexane/ethyl acetate, 9:1), yielding1.910 g of compound (94%) as a colourless oil. - [α]_(D) ²²=−3.61(c=2.52, CHCl₃). - ¹H NMR (200 MHz, CDCl₃): δ=−0.5 (s, 6 H,CH₃Si), 0.85[s, 9 H, (CH₃)₃C—Si], 1.4 [s, 9 H, C(CH₃)₃], 1.5-2.1 (m, 4 H, CH₂—CH₂),2.9 (m, 1 H, SiO—CH₂—CH—N), 3.3-3.4 (m, 3 H, N—CH—COOtBu, SiO—CH₂), 3.9(s, 2 H, CH₂Ph), 7.3 (m, 5 H, aromatic). - ¹³C NMR (50.3 MHz, CDCl₃):δ=173.6, 139.3, 129.1, 127.9, 126.7, 19.9, 67.5, 66.8, 65.8, 58.8, 28.4,28.0, 27.8, 25.8, 18.1, -3.6.

146. A solution of the silyl protected alcohol (1.850 g, 4.55 mmol) andPd(OH)₂/C 20% (0.250 g, 0.45 mmol) in 45 ml of MeOH was stirred underhydrogen atmosphere for 4 hours. Then the catalyst was filtered throughcelite pad and washed with MeOH, the solvent was evaporated underreduced pressure, yielding 1.34 g of hydrogenated compound (94%) ascolourless oil. - [α]_(D) ²²=−5.80 (c=1.99, CHCl₃). - ¹H NMR (200 MHz,CDCl₃) δ=0.4 (s, 6 H,CH₃Si), 0.92 [s, 9 H, (CH₃)₃C—Si], 1.49 [s, 9 H,C(CH₃)₃], 1.5-2.1 (m, 4 H, CH₂—CH₂), 2.35 (broad, 1 H, NH), 3.2 (m, 1 H,SiO—CH₂—CH—N), 3.65 (m, 3 H, N—CH—COOtBu, SiO—CH₂).

147. To a stirred solution of the previous compound (1.2 g, 3.79 mmol)in 38 mil of CH₂Cl₂ were added pyridine (11.39 mmol, 0.92 ml) and(CF₃CO)₂O (8.35 mmol, 1.16 ml). After 1.5 hours the solvent wasevaporated under reduced pressure and the crude purified by flashchromatography (hexane/ethyl acetate, 9:1), yielding 1.4 g of theN-protected pyrrolidine (89%) as colourless oil. - [α]_(D) ²²=−8.62(c=2.11, CHCl₃). - ¹H NMR (200 MHz, CDCl₃): 8 0.4 (s, 6 H,CH₃Si), 0.9[s, 9 H, (CH₃)₃C—Si], 1.47 [s, 9 H, C(CH₃)₃], 1.7-2.4 (m, 4 H, CH₂—CH₂),3.5 (m, 1 H, SiO—CHH), 3.75 (dd, J=10.6 Hz, J=4.2 Hz, 1 H, SiO—CHH), 4.2(m, 1 H, SiO—CH₂—CH—N), 4.35 (t, J=8.5 Hz 1 H, N—CH—COOtBu).

148. To a stirred solution of N-protected pyrrolidine (1.2 g, 2.91 mmol)in 29 ml of THF, cooled at −40° C., was added a 1M solution of TBAF inTHE (3.20 mmol, 3.2 ml). Then the solution was allowed to warm at roomtemp. After 2.5 hours was added 30 ml of brine and the resulting mixturewas extracted with ethyl acetate. The organic phase was dried withNa₂SO₄ and the solvent evaporated under reduced pressure. The crude waspurified by flash chromatography (hexane/ethyl acetate, 6:4), yielding0.850 g of O-deprotected compound (98%) as colourless oil. - [α]_(D)²²=−6.40 (c=1.45, CHCl₃). - ¹H NMR (200 MHz, CDCl₃): δ=1.5 [s, 9 H,C(CH₃)₃], 2.0-2.4 (m, 4 H, CH₂—CH₂), 3.4-3.7 (m, 2 H, HO—CH₂), 4.2-4.6(m, 3 H, N—CH—COOtBu, HO—CH₂—CH—N).

149. The general procedure D was followed using the alcohol and theresidue was purified by flash chromatography (hexane/ethyl acetate,6:4), yielding the aldehyde (93%) as white solid. - [α]_(D) ²²=22.48(c=1.53, CHCl₃). - ¹H NMR (200 MHz, CDCl₃): δ=1.5 [s, 9 H, C(CH₃)₃],1.8-2.5 (m, 4 H, CH₂—CH₂), 4.5-4.7 (m, 2 H, CHO—CH—N, N—CH—COOtBu), 9.7(s, 1 H, CHO).

Enamide (40)

150. The general procedure A was followed using 13 and the crude residuewas purified by flash chromatography affording the enamide (68%) ascolourless oil (diastereoisomeric ratio Z:E=1:1). ¹H NMR (200 MHz,CDCl₃) (signals were splitted for amidic isomerism and were referred tothe mixture of two diastereoisomers): δ=1.5 [s, 9 H, C(CH₃)₃], 1.6-2.45(m, 4 H, CH₂—CH₂), 3.75 (s, 3 H, COOCH₃), 4.6 (m, 1 H, N—CH—COOtBu), 4.8(dd, J=18 Hz, J=10 Hz, 1 H, ═CH—CH—N), 5.12 (s, 2 H, CH₂Ph), 6.3, 6.8(2d, J=10 Hz, 1 H, ═CH of Z-isomer, E-isomer), 7.35 (m, 5 H, aromatic.

151. The general procedure B was followed using the enamide and thecrude was purified by flash chromatography affording 40 with a 95% ofyield as colourless oil. - ¹H NMR (200 MHz, C₆D₆) (signals were splittedfor amidic isomerism and were referred to the mixture of twodiastereoisomers): δ=1.3, 1.5 [2 s, 18 H, C(CH₃)₃], 1.6-2.35 (m, 4 H,CH₂—CH₂), 3.7 (s, 3 H, COOCHI₃), 4.6-4.8 (m, 2 H, N—CH—COOtBu,—CH—CH—N), 5.25 (m, 2 H, CH₂Ph), 7.0 (m, 1 H, ═CH), 7.35 (m, 5 H,aromatic). ¹³C NMR (50.3 MHz, C₆D₆) (signals were splitted for amidicisomerism and were referred to the mixture of two diastereoisomers):δ=169.1, 163.9, 141.2, 136.1, 129.9, 128.4, 128.2, 127.4, 119.4, 113.7,83.6, 82.5, 82.0, 68.8, 68.5, 68.2, 62.5, 60.9, 60.8, 58.5, 57.6, 56.8,53.2, 51.9, 51.7, 51.6, 33.7, 31.8, 30.2, 27.7, 27.5, 26.9.

Aminoester (41)

152. A Z/E mixture of 40 (0.609 g, 1.01 mmol) and Pd(OH)₂/C 20% (0.054g) in 10 ml of MeOH was stirred under hydrogen atmosphere for 18 h. Thecatalyst was filtered through a celite pad and washed with MeOH. Thesolvent was evaporated under reduced pressure and the crude purified byflash chromatography (toluene/Et₂O, 85:15), yielding 0.365 g of 40 (77%)as yellow oil. - ¹H NMR (200 MHz, CDCl₃) (signals were splitted foramidic isomerism and were referred to the mixture of twodiastereoisomers): δ=1.45 [s, 18 H, C(CH₃)₃], 1.6-2.7 (m, 6 H, CH₂—CH₂,BocN—CH—CH₂), 3.75 (2 S, 3 H, COOCH₃), 4.25-4.4 (2 m, 2 H, BocN—CH,BocN—CH—CH₂—CH), 4.55 (m, 1 H, N—CH—COOtBu), 5.30 (d, J=8.5 Hz, 1 H,NH). -¹³C NMR (50.3 MHz, CDCl₃) (signals were splitted for amidicisomerism and were referred to the mixture of two diastereoisomers):δ=172.4, 170.0, 155.8, 128.9, 128.0, 82.7, 82.0, 79.7, 61.4, 60.6, 58.0,56.5, 52.2, 51.5, 37.7, 36.4, 35.5, 30.2, 29.7, 29.0, 28.4, 28.1, 27.6,25.5. - FAB⁺MS: calcd, for C₂₀H₃₁F₃N₂O₇468.47, found 468.

Amino acid (42)

153. A solution of 41 (0.184 g, 0.393 mmol) and NaBH₄ (0.0298 g, 0.781mmol) in 8 ml of MeOH was stirred for 1 hour at room temperature. Thesolution was concentrated and 10 ml of water was added. The aqueoussolution was extracted with ethyl acetate, the collected organic phaseswere dried on Na₂SO₄ and the solvent evaporated under reduced pressure.The two diastereoisomers formed in the previous reactions were separatedat this step by flash chromatography (ethyl acetate/hexane, 6:4),achieving 0.123 g of 42 (R) and 42 (S) (84%) in a 2.6:1diastereoisomeric ratio as colourless oil. - 42 (R): - ¹H NMR (200 MHz,C₆D₆) (signals were splitted for amidic isomerism): δ=1.30, 1.45 [2 s,18 H, C(CH₃)₃], 1.5-1.9 (m, 6 H, CH₂—CH₂, BocN—CH—CH₂), 2.85 (m, 1 H,BocN—CH—CH₂—CH), 3.2-3.4 (m, 4 H, COOCH₃, N—CH—COOtBu), 4.65 (m, 1 H,3BocN—CH), 6.6 (broad, 1 H, NHBoc). - ¹³C NMR (50.3 MHz, C₆D₆) (signalswere splitted for amidic isomerism): δ=174.1, 173.2, 155.8, 81.4, 81.3,79.5, 60.6, 60.4, 56.5, 56.3, 52.5, 52.0, 37.7, 31.9, 30.0, 29.8, 28.2,28.0, 27.9. - FAB⁺MS: calcd. for C₁₈H₃₂N₂O₆ 372.46, found 373. - 42(S): - 1H NMR (200 MHz, C₆D₆) (signals were splitted for amidicisomerism): δ=1.30, 1.50 [2 s, 18 H, C(CH₃)₃], 1.50-1.80 (m, 6 H,CH₂—CH₂, BocN—CH—CH₂), 2.8 (m, 1 H, BocN—CH—CH₂—CH), 3.3 (s, 3 H,COOCH₃), 3.4 (dd, J=9.1 Hz, J=5.9 Hz, 1 H, N—CH—COOtBu), 4.45 (m, 1 H,BocN—CH), 5.3 (broad, 1 H, NHBoc). - ¹³C NMR (50.3 MHz, C₆D₆) (signalswere splitted for amidic isomerism): δ=171.7, 171.5, 164.2, 164.0,154.7, 154.3, 153.5, 136.6, 136.4, 135.8, 128.4, 128.3, 128.2, 128.1,127.7, 126.2, 125.9, 125.8, 81.0, 87.1, 66.8, 66.6, 60.8, 60.4, 58.2,57.5, 52.3, 52.2, 32.8, 31.9, 28.5, 28.1, 27.8, 27.7, 27.4, 27.1. -FAB⁺MS: calcd. for C₁₈H₃₂N₂O₆ 372.46, found 373.

EXAMPLE 9 5,5-Fused bicyclic lactam [1a]

154. A stirred solution of 42 (S) (0.028 g, 0.075 mmol) in 1.5 ml ofp-xylene was warmed at 130° C. for 24 hours. The solvent was thenevaporated under reduced pressure and the crude purified by flashchromatography (hexane/ethyl acetate, 7:3), yielding 19 mg of 1a (74%)as a white foam. - [α]_(D) ²²=−4.80 (c=1.20, CHCl₃). - ¹H NMR (200 MHz,CDCl₃): δ=1.50, 1.51 [2 s, 18 H, C(CH₃)₃], 1.6-2.4 (m, 5 H, CH₂—CH₂,BocN—CH—CHH), 2.95 (m, 1 H, BocN—CH—CHH), 3.85 [m, 1 H, (CH—N], 4.15 (d,J=8.8 Hz, 1 H, N—CH—COOtBu), 4.60 (m 1 H, CH—NBoc), 5.25 (broad, 1 H,NH). - ¹³C NMR (50.3 MHz, CDCl₃) (signals were splitted for amidicisomerism); δ=171.7, 169.7, 155.6, 81.8, 79.5, 58.8, 56.5, 56.0, 55.8,39.5, 33.4, 29.5, 28.2, 27.8. - FAB⁺MS: calcd. for C₁₇H₂₈N₂O₅ 340.41,found 341.

EXAMPLE 10 5,5-Fused bicyclic lactam [7a]

155. The compound [7a] was achieved from compound 42 (R), by using thesame procedure described for the synthesis of compound 1a, with a 65% ofyield as white foam. - [α]_(Dhu 22)=−4.80 (c=1.20, CHCl₃). - ¹H NMR (200MHz, CDCl₃): δ=1.45 [2 s, 18 H, C(CH₃)₃], 1.5-2.5 (m, 6 H, CH₂—CH₂,BocN—CH—CH₂), 4.05 (d, J=8.8 Hz, 1 H, N—CH—COOtBu), 4.12 (m, 1 H, CH—N),4.25 (m, 1 H, CH—NBoc), 5.05 (broad, 1H, NH). - ¹³C NMR (50.3 MHz,CDCl₃) (signals were splitted for amidic isomerism): δ=170.9, 169.8,155.2, 82.2, 81.8, 79.9, 77.1, 61.2, 58.8, 57.6, 56.0, 55.8, 34.4, 33.8,33.4, 29.9, 29.5, 29.2, 28.5, 28.1, 27.7. - FAB⁺MS: calcd. forC₁₇H₂₈N₂O₅ 340.41, found 341.

Aldehyde (14, 17)

156. To a stirred solution of 43 (1.205 g, 3.08 mmol) in drydiethylether (31 mL) at −10° C., LiBH₄ 2M in THF (1.5 mL, 3.08 mmol) wasadded. After 24 h a saturated solution of NaHCO₃ (40 ml) was added andthe resulting mixture was extracted with AcOEt. The organic phase wasdried over Na₂SO₄ and evaporated to dryness. The crude product waspurified by flash chromatography (hexanelethyl acetate 1:1), yielding1.01 g of alcohol (94%) as a yellow oil. - Trans-isomer: [α]_(D)²²=−32.3 (c=1.02, CHCl₃).- ¹H NMR (200 MHz, CDCl₃): δ=1.35 [s, 9 H,C(CH₃)₃], 1.5-2.4 (m, 6 H, CH₂—CH₂, CH₂—CH₂—O), 3.5-3.7 (m, 2 H, CH₂OH),3.82 (bs, 1 H, OH), 4.22 (dd, J=7.5, J˜0, 1 H, CHCO₂tBu), 4.38 (m, 1 H,CH₂—CH—N), 5.15 (m, 2 H, CH₂Ph), 7.32 (s, 5 H, aromatic). - ¹³C NMR(50.3 MHz, CDCl₃) (signals were splitted for amidic isomerism): δ=171.4,156.1, 136.0, 128.4, 128.3, 127.9, 127.8, 127.7, 81.2, 81.1, 67.2, 67.0,60.4, 59.9, 59.0, 55.2, 55.1, 38.6, 37.7, 28.9, 28.7, 27.8, 27.7. -Cis-isomer: [α]_(D) ²²=−54.0 (c=1.51, CHCl₃). - ¹H NMR (200 MHz, CDCl₃):δ=1.33 [s, 9 H, C(CH₃)₃], 1.4-1.24 (m, 6 H, CH₂—CH₂, CH₂—CH₂—O), 3.6-3.9(m, 2 H, CH₂OH), 4.08 (dd, J=9.5, J=4, 1 H, OH), 4.25 (dd, J=J 8.5, 1 H,CHCO₂tBU), 4.40 (m, 1 H, CH₂—CH—N), 5.15 (m, 2 H, CH₂Ph), 7.35 (s, 5 H,aromatic). - ¹³C NMR (50.3 MHz, CDCl₃): δ=27.7, 28.9, 30.4, 37.4, 55.4,58.8, 60.5, 67.4, 81.3, 127.7, 127.9, 128.3, 136.1, 155.9, 171.8.

157. A solution of the alcohol (0.304 g, 0.87 mrnol) in dry CH₂Cl₂ (2.5mL) was added to a Suspension of Dess-Martin periodinane (0.408 g, 1.13mmol) in dry CH₂Cl₂ (2-5 mL) at room temperature. After 1h Et₂O and NaOH1N were added till clear solution. The aqueous phase was extracted twicewith Et₂O; the collected organic layers were washed with H₂O, dried withNa₂SO₄, and evaporated to dryness. The crude product was purified byflash chromatography (hexane/ethyl acetate 7:3) affording 0.277 g of 17(92%). - Tranis-isomer: [α]_(D) ²²=−48.65 (c=1.01, CHCl₃). - ¹H NMR (200MHz, CDCl₃) (signals were splitted for amidic isomerism): δ=1.35-1.45 [2s, 9 H, C(CH₃)₃], 1.6-2.6 (m, 4 H, CH₂—CH₂), 2.8-3.1 (2 m, 2 H, CH₂CHO),4.3 (m, 1 H, CHO—CH₂—CH—N), 4.6 (m, 1 H, N—CH—COOR), 5.15 (m, 2 H,CH₂Ph), 7.30 (m, 5 H, aromatic), 9.1, 9.3 (2 m, IH, CHO). - ¹³C NMR(50.3 MHz, CDCl₃) (signals were splitted for amidic isomerism): δ=200.3,171.4, 154.1, 136.2, 128.4, 128.2 128.0, 127.8, 127.7, 81.3, 67.1, 66.9,60.5, 60.1, 53.4, 52.5, 49.0, 48.4, 29.5, 28.6, 28.3, 27.8, 27.7, 27.3.

158. N-Boc-protected enamide (44): The mixture of aldehydes 14 and 17was reacted following the general procedure A. The crude product waspurified by flash chromatography (hexane/ethyl acetate 7:3), affordingthe enamide in 99% yield, as a trans:cis, Z/E mixture. Trans-Z-isomer:[α]_(D) ²²=−61.84 (c=1.01, CHCl₃). - ¹H NMR (200 MHz, CDCl₃) (signalswere splitted for amidic isomerism): δ=1.35-1.50 [2 s, 9 H. C(CH₃)₃],1.6-2.3 (m, 4 H, CH₂—CH₂), 2.3-2.8 (2 m, 2 H, ═CH—CH₂), 3.75 (a, 3 H,COOCH₃), 4.15-4.25 (2 m, 2 H, —CH₂—CH—N and N—CH—COOtEu), 5.15 (m, 4 H,CH₂Ph), 6.55 (t, J=8.5 Hz, 1 H, ═CH), 7.35 (m, 10 H. aromatic). - ¹³CNMR (50.3 MHz, CDCl₃) (signals were splitted for amidic isomenrism):δ=171.4, 164.8, 164.6, 154.4, 153.9, 153.7, 136.4, 136.2, 135.9, 135.7,133.0, 132.0, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.6,126.7, 81.2, 67.3, 67.2, 67.0, 66.8, 60.6, 60.2, 57.6, 56.7, 52.3, 33.5,32.5, 28.5, 27.7, 27.4. - FAB⁺MS: calcd. for C₃₀H₃₆N₂O₈ 552.6, found552. -

159. Trans-E-isomer [α]_(D) ²²=−50.16 (c=1.48, CHCl₃). - ¹H NMR (200MHz, CDCl₃) (signals were splitted for amidic isomerism): δ=1.35-1.45 [2s, 9 H, C(CH₃)₃], 1.6-2.4 (m, 4 H, is CH₂—CH₂), 2.7-3.1 (2 m, 2 H,═CH—CH₂), 3.8 (2 s, 3 H, COOCH₃) 4.1-4.3 (2 m, 2 H, —CH₂—CH—N eN—CH—COOtBu), 5.10 (m, 4 H, CH₂Ph), 6.50 (m, 1 H, ═CH), 7.25 (m, 10 H,aromatic). - ¹³C NMR (50.3 MHz, CDCl₃) (signals were splitted for amidicisomerism): δ=171.7, 171.5, 164.2, 164.0, 154.7, 154.3, 153.5, 136.6,136.4, 135.8, 128.4, 128.3, 128.2, 128.1, 127.7, 126.2, 125.9, 125.8,81.0, 87.1, 66.8, 66.6, 60.8, 60.4, 58.2, 57.3, 52.2, 32.8, 31.9, 28.5,28.1, 27.8, 27.7, 27.4, 27.1.

160. The mixture of enamides (0.394 g, 0.71 mmol) was reacted followingthe general procedure B. Flash chromatography of the crude product(hexane/ethyl acetate 75:25) afforded 0.287 g (73%) of pure trans-isomer23. - Z-isomer: [α]_(D) ²²=−50.98 (c=1.56, CHCl₃). - ¹H NMR (200 MHz,CDCl₃) (signals were splitted for amidic isomerism): δ=1.3-1.5 [4 s, 18H, C(CH₃)₃], 1.7-2.6 (m, 6 H. CH₂—CH₂ and ═CH—CH₂), 3.7 (s, 3 H,COOCH₃), 4.1-4.3 (m, 2 H, —CH₂—CH—N and N—CH—COOtBu), 5.15 (m, 4 H,CH₂Ph), 6.8 (m, 1 H, ═CH), 7.30 (m, 10 H, aromatic). - ¹³C NMR (50.3MHz, CDCl₃) (signals were splitted for amidic isomerism). δ=171.4,163.9, 154.6, 154.5, 150.0, 146.2, 138.5, 138.0, 136.2, 129.9, 128.3,128.2, 128.1, 127.8, 83.4, 81.2, 68.3, 67.0, 66.8, 60.6, 60.2, 56.9,56.2, 52.2, 32.9, 32.0, 28.3, 27.8, 27.7, 27.3. - FAB⁺MS: calcd. forC₃₅H₄₄N₂O₁₀ 652.7, found 652.

161. E-isomer: ¹H NMR (200 MHz, CDCl₃): δ=1.3-1.4 [2 s, 18 H, C(CH₃)₃],1.5-2.3 (m, 4 H, CH₂—CH₂), 3.0 (2 m, 2 H, ═CH—CH₂), 3.65 (2 s, 3 H,COOCH₃), 4.2 (m, 2 H, —CH₂—CH—N and N—CH—COOtBu), 5.15 (m, 4 H, CH₂Ph),6.1 (2 t, J=8.5 Hz, 1 H, ═CH), 7.30 (m, 10 H, aromatic). - ¹³C NMR (50.3MHz, CDCl₃): δ=171.5, 163.7, 154.6, 154.3, 152.2, 150.4, 142.7, 142.2,136.3, 135.1, 128.9, 128.3, 128.2, 128.0, 127.8, 127.7, 83.4, 83.3,81.1, 77.1, 68.3, 66.9, 66.7, 60.7, 60.3, 57.6, 56.8, 51.7, 32.9, 32.0,28.4, 28.0, 27.7, 27.3, 27.0.

EXAMPLE 11 6,5 fused bicyclic lactams (5a, 11a)

162. A solution of 44 (0.489 g, 0.75 mmol) and Pd(OH)₂/C 20% (catalytic)in MeOH (7.5 mL) was stirred under H₂ for one night. The catalyst wasfiltered off and the mixture was refluxed for 24h. The solvent was thenremoved and the two diastereoisomeric products were separated by flashchromatography (hexane/ethyl acetate 6:4), yielding 0.186 g of 5a and11a (70%) in a 1.4:1 diastereoisomeric ratio. - 5a: ¹H NMR (200MHz,CDCl₃). δ=1.45-1.50 [2 s, 18 H, C(CH₃)₃], 1.55-2.60 (m, 8 H, CH₂—CH₂ andBocN—CH—CH₂—CH₂), 3.68 [tt, J=14.9 Hz and 4.2 Hz, 1 H, (R)₂CH—N], 4.05(m, 1 H, CH—NBoc), 4.35 (t, J=8.5 Hz, 1H, N—CH—COOtBu), 5.28 (broad, 1H, NH). - FAB⁺MS. calcd. for C₁₈H₃₂N₂O₅ 354.46, found 354.

163. 11a: [α]_(D) ²²=−107.9 (c=1.7, CHCl₃). ¹H NMR (200 MHz, CDCl₃):δ=1.45-1.50 [2 s, 18 H, C(CH₃)₃], 1.75-2.50 (m, 8 H, CH₂—CH₂ andBocN—CH—CH₂—CH₂), 3.70 [m, 1 H, CH—N], 4.15 (m, 1 H, CH—NBoc), 4.50 (t,J=7.0 Hz, 1H, N—CH—COOtBu), 5.55 (broad, 1 H, NH). - ¹³C NMR (50.3 MHz,CDCl₃): δ=170.6, 168.5, 155.5, 81.4, 79.3, 59.0, 56.2, 49.9, 32.3, 28.1,27.8, 26.5, 25.9. - FAB⁺MS: calcd. for C₁₈H₃₂N₂O₅ 354.46, found 354.

Aldehyde (18)

164. The general procedure C was followed using 43 and the crude residuewas purified by flash chromatography affording the alcohol with a yieldof 98%. ¹H NMR (200 MHz, CDCl₃) δ=1.32 [s, 9 H, C(CH₃)₃], 1.4-2.4 (m, 8H, CH₂—CH₂), 3.5-3.7 (m, 2 H, CH₂OH), 4.1 (m, 1 H, CH₂—CH—N), 4.24 (m, 1H, N—CH—COOtBu), 5.05 (s, 2 H, CH₂Ph), 7.25 (m, 5 H, aromatic).

165. The general procedure D was followed using the alcohol and thecrude was purified by flash chromatography (hexane/ethyl acetate 6:4)affording 18 with a yield of 82% - ¹H NMR (200 MHz, CDCl₃), (signalswere splitted for amidic isomerism): δ=1.32, 1.45 [2 s, 9 H, C(CH₃)₃],1.5-2.7 (m, 8 H, CH₂—CH₂), 4.1 (m, 1 H, CH₂—CH—N), 4.25 (m, 1 H,N—CH—COOR), 5.15 (s, 2 H, CH₂Ph), 7.20-7.40 (m, 5 H, aromatic), 9.6-9.8(2 m, 1 H, CHO),

Enamide (46)

166. The general procedure A was followed using 18 and the crude waspurified by flash chromatography (hexane/ethyl acetate 6:4) affordingthe enamide with a yield of 90% (diastereomeric ratio Z/E=7:1) - ¹H NMR(200 MHz, CDCl₃), (signals were splitted for amidic isomerism): δ=1.32,1.42 [s, 9 H, C(CH₃)₃], 1.5-2.7 (m, 8 H, CH₂—CH₂), 3.71 (s, 1 H,COOCH₃), 4.1 (m, 1 H, CH₂—CH—N), 4.22 (m, 1 H, N—CH—COOtBu), 5.0-5.20(m, 4 H, CH₂Ph), 6.6 (m, 1 H, ═CH), 7.20-7.45 (m, 10 H, aromatic).

167. The general procedure B was followed using the enamide and thecrude residue was purified by flash chromatography yielding 46 (98%). -¹H NMR (200 MHz, CDCl₃), (signals were splitted for amidic isomerism):δ=1.32, 1.42 [2 s, 18 H, C(CH₃)₃], 1.5-2.2 (m, 8 H, CH₂—CH₂), 3.71 (s, 1H, COOCH₃), 3.9 (m, 1 H, CH₂—CH—N), 4.22 (m, 1 H, N—CH—COOtBu), 5.0-5.20(m, 4 H, CH₂Ph), 6.9 (m, 1 H, ═CH), 7.20-7.45 (m, 10 H, aromatic). - ¹³CNMR (50.3 MHz, CDCl₃) (signals were splitted for amidic isomerism):δ=141.6, 128.4, 128.2, 128.1, 127.8, 127.7, 68.2, 66.8, 60.5, 58.1,52.1, 31.3, 29.5, 27.1, 27.3, 24.6.

EXAMPLE 12 trans-7,5-fused bicyclic laetam (6a, 12a)

168. To a solution of 46 (0.093 g, 0.141 rnmol) in MeOH (2 ml) was added1N NaOH (0.705 mmol, 0.705 ml) and stirred for 1.5 h. The solution wasacidified until pH 3 with 1N HCl, then the solution was evaporated. Thecrude was submitted to the next reaction without further purification. -¹H NMR (200 MHz, CDCl₃) (signals were splitted for amidic isomerism):δ=1.25, 1.48 [2 s, 18 H, C(CH₃)₃], 1.5-2.4 (m, 8 H, CH₂—CH₂), 4.1 (m, 1H, CH₂—CH—N), 4.3 (m, 1 H, N—CH—COOtBu), 5.12 (s, 2 H, CH₂Ph), 6.65 (m,1 H, ═CH), 7.1-7.4 (m, 5 H, aromatic), 9.00 (bs, 1 H, —COOH).

169. A solution of previous compound in xylene was refluxed for 48 h.The solvent was evaporated and the crude was purified by flashchromatography yielding 6a and 12a with a 40% of yield.

170. 6a - ¹H NMR (200 MHz, CDCl₃) (signals were splitted for amidicisomerism): δ=1.43, 1.45 [2 s, 18 H, C(CH₃)₃], 1.51-2.40 (m, 10 H,CH₂—CH₂), 3.75 [m, 1 H, CH—N], 4.22 (m, 1 H, CH—NBoc), 4.48 (t, J=17 Hz,1H, N—CH—COOtBu), 5.7 (broad, 1 H, NH).

171. 12a - ¹H NMR (200 MHz, CDCl₃) (signals were splitted for amidicisomerism): δ=1.47, 1.48 [2 s, 18 H, C(CH₃)₃], 1.55-2.50 (m, 8 H,CH₂—CH₂), 4.0 (m, 1 H, CH—N), 4.30 (m, 1 H, CH—NBoc), 4.50 (dd, J=5.4Hz, J=17 Hz, 1H, N—CH—COOtBu), 6.0 (bd, 1 H, NH).

EXAMPLE 13

172. Using the bicyclic lactams prepared according to the precedingexamples, the respective peptidomimetics compounds, containing the RGDsequence were prepared according to the method disclosed in Gennari etal.: Eur. J. Org. Chem., 1999, 379-388.

1. Compounds of formula (I)

wherein n is the number 0, 1 or 2, Arg is the amino acid L-Arginine, Glyis the amino acid Glycine and Asp is the amino acid L-Aspartic acid andthe pharmaceutically acceptable salts thereof, their racemates, singleenantiomers and diastereoisomers.
 2. A compound of claim 1 which is


3. A compound of claim 1 , which is


4. A compound of claim 1 , which is


5. A compound of claim 1 , which is


6. A compound of claim 1 , which is


7. A compound of claim 1 , which is


8. A compound of claim 1 , which is


9. A compound of claim 1 , which is


10. A process for the preparation of the compounds of claim 1 comprisingthe following steps: a) Horner-Emmons olefination of a compounld offormula (II)

wherein R is a lower alkyl residue; R₁ is a suitable nitrogen protectinggroup, to give a compound of formula (III);

wherein R₃ is a suitable nitrogen protecting group, R₄ is a lower alkylresidue; b) hydrogenation of said compound of formula (III) andcyclisation; and, if desired c) separation of the stereoisomericmixture; d) building of the RGD cyclic sequence; and, if desired e)separation of the stereoisomeric mixture.
 11. A process for thestereoselective synthesis of the compounds of claim 1 , comprising thefollowing steps: a) Horner-Emmons olefmation of a compound of formula(II)

wherein R is a lower alkyl residue; R₁ is a suitable nitrogen protectinggroup, to give a compound of formula (III);

wherein R₃ is a suitable nitrogen protecting group, R₄ is a lower alkylresidue; b) hydrogenation of said compound of formula (III) by chiralphosphine-Rh catalysed hydrogenation and cyclisation; and, if desired c)separation of the stereoisomeric mixture; d) building of the RGD cyclicsequence; and, if desired e) separation of the stereoisomeric mixture.12. Pharmaceutical composition comprising a therapeutically orpreventive effective dose of at least a compound of claim 1 in admixturewith pharmaceutically acceptable vehicles and/or excipients.
 13. Amethod for selectively inhibiting α_(v)β₃ integrin-mediated cellattachment to an RGD-containing ligand, comprising contacting saidligand with an effective amount of a compound of claim 1 .
 14. A methodfor treating a subject suffering from a pathology related to an alteredα_(v)β₃ integnin-mediated cell attachment comprising administering tosaid subject a compound of claim 1 .
 15. A method according to claim 14, wherein said pathology is retinopathy.
 16. A method according to claim14 , wherein said pathology is acute renal failure.
 17. A methodaccording to claim 14 , wherein said pathology is osteoporosis.
 18. Amethod for treating a subject suffering from altered angiogenesis,comprising administering to said subject a compound of claim 1 .
 19. Amethod for the treatment of tumors in a subject comprising administeringto said subject a compound of claim 1 .
 20. A method according to claim19 , wherein said tumor, is associated with metastasis.